Methods of inhibiting hiv infections by pre-exposure prophylaxis

ABSTRACT

A process is provided for protecting a primate host from a self-replicating infection by an immunodeficiency retrovirus. Protection is achieved by administering to the primate host a combination of a pharmaceutically effective amount of a nucleoside reverse transcriptase inhibitor and a pharmaceutically effective amount of a nucleotide reverse transcriptase inhibitor prior to exposure to the immunodeficiency retrovirus. The administration is effective if provided in a single dose within 24 hours of the exposure. A regime of regular daily doses is also effective in providing protection against an immunodeficiency retrovirus becoming self-replicating after infecting a primate host. A process for controlling retrovirus transmission within a population includes the administration to a subpopulation at high risk for contracting an immunodeficiency retroviral infection the detailed combination prior to sexual exposure to a source of immunodeficiency retrovirus so as to preclude the immunodeficiency retrovirus from becoming self-replicating in a member of the subpopulation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 16/413,381,filed on May 15, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/913,750,filed on Mar. 6, 2018, issued as U.S.Pat. No. 10,335,423, which is a continuation of U.S. patent applicationSer. No. 15/406,344, filed on Jan. 13, 2017, issued as U.S. Pat. No.9,937,191, which is a continuation of U.S. patent application Ser. No.14/679,887, filed on Apr. 6, 2015, issued as U.S. Pat. No. 9,579,333,which is a continuation of U.S. patent application Ser. No. 11/669,547,filed on Jan. 31, 2007, issued as U.S. Pat. No. 9,044,509, which claimsthe benefit of U.S. provisional application 60/764,811, filed on Feb. 3,2006. All of the prior applications are incorporated herein by referencein their entirety.

GOVERNMENT INTEREST

The invention described herein may be manufactured, used, and licensedby or for the United States Government.

FIELD OF THE INVENTION

The present invention in general relates to a process for inhibitinginitial infection by a retrovirus such as human immunodeficiency virus(HIV) and in particular to a combination of a nucleoside reversetranscriptase inhibitor (NRTI) and a nucleotide reverse transcriptaseinhibitor (NtRTI) capable of preventing self-replicating retroviralinfection, even in response to multiple viral challenges.

BACKGROUND OF THE INVENTION

Despite the fact that significant progress has been made slowing theadvancement of the symptoms of AIDS associated with HIV infection, inthe absence of an effective vaccine, HIV continues to spread globally.The spread of HIV persists in part because an infected individualremains a potential source of injection. It is clear that currenttreatment of monitoring viral titer and in response to a titer exceedinga preselected threshold commencing treatment with highly activeantiretroviral therapy (HAART) has not prevented new infections.

An attractive method of controlling the spread of HIV would be toprovide an individual exposed to a potential source of HIV with apre-exposure prophylactic treatment. As HIV and, in particular HIV-1,often begins with a comparatively small population of retroviralparticles being transmitted to a new host and within a few daysself-replicating into a retroviral titer detectable in host blood serum.If the establishment of a retroviral could be blocked before the HIVburden expands into a self-propagating infection, an individual couldavoid contraction of HIV.

Previous attempts at pre-exposure prophylaxis have met with limitedsuccess. Prophylactic activity has been demonstrated with the NtRTI,tenofovir in monkey models challenged with simian immunodeficiency virus(SIV).¹⁻³ Unfortunately, oral daily dosing and pre-exposure prophylaxiswith tenofovir at a dose equivalent to that used in humans proved toonly be partially protective against rectal SHIV transmission.⁴

HAART therapy involves the administration of a combination including atleast three active compounds classified by the mode of operation as anNRTI, an NtRTIs, a non-nucleoside reverse transcriptase inhibitors(NNRTIs), protease inhibitor, and an entry inhibitor. While HAART iseffective in lowering retroviral titer in a host, concerns remain as tothe long term toxicity and the retained potential to infect others. Itis also unknown if initiating HAART therapy in a pre-exposureprophylactic regimen would be efficacious. As a result, society remainsdevoid of a pre-exposure prophylactic regimen to prevent an individualfrom developing self-propagating retrovirus infection subsequent toinitial exposure.

Thus, there exists a need for a chemoprophylactic composition and dosingregimen effective in blocking early stage infection by retrovirus in ahost founder cell population. There also exists a need for achemoprophylactic composition formulated with a vehicle amenable to usercompliance.

SUMMARY OF THE INVENTION

A process is provided for protecting a primate host from aself-replicating infection by an immunodeficiency retrovirus. Protectionis achieved by administering to the primate host a combination of apharmaceutically effective amount of a nucleoside reverse transcriptaseinhibitor and a pharmaceutically effective amount of a nucleotidereverse transcriptase inhibitor prior to exposure to theimmunodeficiency retrovirus. The administration is effective if providedin a single dose prior to the exposure. A regime of multiple temporallyspaced doses prior to retroviral exposure is also effective in providingprotection against an immunodeficiency retrovirus becomingself-replicating after infecting a primate host. A process forcontrolling retrovirus transmission within a population includes theadministration to a subpopulation at high risk for contracting animmunodeficiency retroviral infection a combination of apharmaceutically effective nucleoside reverse transcriptase inhibitorand a pharmaceutically effective amount of a nucleotide reversetranscriptase inhibitor prior to exposure to a source ofimmunodeficiency retrovirus so as to preclude the immunodeficiencyretrovirus from becoming self-replicating in a member of thesubpopulation.

A kit is also provided that includes at least one combination dose of apharmaceutically effective amount of a nucleoside reverse transcriptaseinhibitor and a pharmaceutically effective amount of a nucleotidereverse transcriptase inhibitor sufficient to protect a primate hostfrom developing a self-replicating retroviral infection along withinstructions for the administration of the at least one dose one priorto and optionally one additional dose subsequent to a potential exposureto an immunodeficiency retrovirus along with dosing modificationsassociated with subject characteristics and behaviors to further reducethe risk of contracting a self-replicating immunodeficiency retrovirusinfection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depicting one study of the present invention for 4groups of macaques in which all treated macaques received knownantiretroviral medications 7 to 9 days prior to the first virusinoculation and continuing throughout the study with treated animalsthat remained uninfected throughout the 14 viral challenges receiving 28additional days of post-exposure prophylactics.

FIG. 2 is a survival curve graph for macaque Groups 1-4 per FIG. 1, aswell as for animals receiving only tenofovir disoproxil fumarate (TDF).

FIG. 3 is a graph depicting a plot of viremia as a function of time foruntreated controls (∘) and breakthrough infections (●) where each pointrepresents a mean viremia observed, 0 time indicates peak plasma virusload observed in a given animal where the arrow bars denote standarderror of the mean (SEM).

FIG. 4 depicts plots of infection dynamics as a function of time duringthe study per FIG. 1 with plots for animals coded as AG-80, AG-46, AH-04and AG-07 corresponding to emtricitabine (FTC) treatment alone, or FTCplus TDF treatment (AI-54 and AG-81). The arrow indicates the firstdetectable antibody response. Grey circles indicate detectable M184V/Imutation; wild type sequences are shown in as black full circles. Opencircles indicate the time points where no genotype was undertaken.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention has utility in protecting a primate host fromself-propagating immunodeficiency virus infection. The use of acombination of antiretroviral agents as a prophylactic dosing regime isalso provided for the manufacture of a medicament is provided forprotection against a human immunodeficiency virus infection developingto a level of self-replicating infection. Retroviral transmissionthrough most routes entails a new primate host receiving a small numberof viral particles. Common routes of retrovirus transmissionillustratively include sexual intercourse, medical worker skin punctureinoculation, hypodermic needle sharing, blood transfusions, birth canalexposure, breastfeeding, and transplacental contact between individuals.Through the administration of at least one nucleoside reversetranscriptase inhibitor (NRTI) and at least one nucleotide reversetranscriptase inhibitor (NtRTI) prior to a retrovirus exposureprotection is provided against development of a self-replicatingretroviral infection. As the aforementioned exposure routes arecharacterized by a small number of retrovirus particles beingtransferred to the new primate host, this initial phase of infectionrepresents a window of opportunity to protect a host from infection. Theinventive chemoprophylactic treatment is provided through a dosingregimen. A dosing regimen according to the present invention thatprovides retroviral protection to a host primate includes at least onesingle dose administered prior to initial retroviral exposure. Aninventive dosing regimen also includes a course of multiple dosesadministered in advance of exposure to maintain a therapeutic level ofNRTI and NtRTI agents in the primate host. The timing of the at leastone does prior to retroviral exposure is dictated by thepharmacokinetics of the NRTI and NtRTI components to assure the presenceof a therapeutically effective amount of inventive composition for atleast 20 hours subsequent to the exposure to the communicated smallretroviral particle population. Multiple doses are administeredaccording to the present invention at regular time intervals and amountssuch as for example like formulated daily doses for a period of severaldays, weeks, or months; or are administered in advance of a likelyexposure as a cluster of doses, with the amount of NRTI and NtRTIcomponents in each dose being independent of the of amount of NRTI andNtRTI in other doses within the cluster. While most oral, topical, andparenteral existing versions of NRTIs and NtRTIs are fully absorbed andtherapeutically active within 1 to 8 hours, it is appreciated thatsubcutaneous implants and long acting timed release formulations allowfor a single dose to sustain therapeutically effective amounts of aninventive prophylactic composition for several days, weeks, or evenmonths. Representative of sustained release compositions and implantsare provided in the U.S. Pat. Nos. 4,122,129; 4,927,687; 4,996,047;5,169,642; and 5,656,296.

The combination of NRTI and NtRTI compounds administeredprophylactically according to the present invention are shown to providea dose-dependent inhibition of HIV self-replicating infection and atherapeutically effective dosing primate host protection againstself-replicating HIV infection is provided, even in response to multipleviral challenges. While the present invention is largely detailed withrespect to HIV-1 as a prototypical infectious and pathogenic retrovirus,it is appreciated that other retroviruses owing to reliance on reversetranscription for replication are also protected against in a primatehost according to the present invention.

As used herein, “protection” as used in the context of a host primateresponse to an immunodeficiency virus challenge is defined by the hostprimate being serologically negative and negative in response to apolymerase chain reaction (PCR) testing for viral genome.

As used herein, the term “retrovirus” is inclusive of any virus thatutilizes reverse transcriptase in the viral replication cycle andtherefore is susceptible to the antiviral activity of nucleoside ornucleotide analogs specifically inclusive of HIV (HIV-1 and HIV-2),HTLV-1, HTLV-2, HTLV-3, HTLV-4, and SIV. Also encompassed are virusessuch as HBV that although not technically classified as retrovirusesnonetheless utilize a reverse transcriptase and are thereforesusceptible to the antiviral activity of nucleoside and/or nucleotideanalogs.

As used herein a “primate host” is defined to include a monkey, baboon,chimpanzee, gorilla, and a human. Nonhuman primates are appreciated tothemselves be susceptible to infection by retroviruses and in particularimmunodeficiency viruses and represent well-established animal models asto human response with an appreciation that physiological differencesoften require different doses in milligrams per kilogram for a nonhumanprimate animal model relative to a human.

The compositions of the present invention include administration incombination of an NRTI and NtRTI and are readily compounded bypharmaceutical composition with conventional pharmaceutically acceptablecarriers or diluents. Additionally, pharmaceutically acceptablederivatives and prodrugs of active NRTIs and NtRTIs operative in thepresent invention include salts such as alkali metal salts; esters suchas acetate, butyrate, octinoate, palmitate, chlorobenzoates, benzoates,C₁-C₆ benzoates, succinates, and mesylate; salts of such esters; andnitrile oxides. It is appreciated that other analogs of pharmaceuticallyactive NRTIs or NtRTIs that provide within a primate host an activeantiviral metabolite residue are also suitable as part of an inventivecomposition. A pharmaceutically acceptable carrier or diluent includesagents that are compatible with other ingredients of a dosage and notinjurious to a primate host. The identity and process for compounding acombination of at least one NRTI and at least one NtRTI into a dosageform suitable for delivery by a route with administration by oral,rectal, topical, vaginal or parenteral routes of administration areprovided in Remington's Science and Practice of Pharmacology, 20^(th)Edition, Chapters 37-47, pages 681-929, where parenteral injectionincludes subcutaneous, intramuscular, intravenous, and intradermalinjection.

As used herein the term “prodrug” is defined to include a compound thatwhen administered to a primate host generates an active NRTI or NtRTI asa result of spontaneous reaction under physiological conditions,enzymatic catalysis, metabolic clearance, or combinations thereof. Anexemplary NtRTI prodrug currently FDA approved for HAART use istenofovir disoproxil fumarate (TDF) and is detailed in U.S. Pat. No.5,935,946.

The present invention provides an alternative to conventional retroviraltherapy using HAART, in response to self-propagating HIV infection byprotecting a primate host against the establishment of self-replicatingretroviral infection that provides an indication for such therapy.Through prophylactic prior dosing with an inventive combinationincluding at least one NRTI and one NtRTI, replication of thecomparatively low number of viral particles received by a host primateis prevented.

To achieve protection against a primate host developing a retroviralself-replicating infection, at least one dosage of an NRTI and NtRTI isadministered to the primate host prior to exposure to the retrovirus.Preferably, the at least one NRTI and at least one NtRTI areadministered concurrently. More preferably, the combination of reversetranscriptase inhibitors is compounded into a single formulation.

The process of the present invention demonstrates protection againstretroviral self-replicating infection through administration of even asingle dosage administered prior to the retroviral exposure. Owing tothe known pK rates of specific NRTIs and NtRTIs, a single dosage isadministered to assure a therapeutically effective amount of NRTI andNtRTI persist in the primate host for a time of more than 12 hours afterviral challenge. With conventional NRTI and NtRTI formulations,currently approved for HAART, preferably an inventive dose isadministered within 12 hours prior to retroviral exposure and still morepreferably often within 2 hours prior to retroviral exposure. Thepractice of the inventive process involving the administration of asingle dosage in the hours proceeding a likely retroviral exposure isparticularly advantageous in assuring compliant dosing in a human andalso avoids side effects associated with a regular dosing regime and isparticularly well suited for a human engaging in a sporadic behaviorlikely to bring the person into retroviral exposure. Preferably, anadditional dose or doses of a combination of at least one NRTI and atleast one NtRTIs is provided subsequent to the retroviral exposure eventto assure adequate antiviral reverse transcriptase inhibitorconcentration during and immediately subsequent to retroviral infectionof the host founder cell population so as to preclude retroviralself-replication to assure NRTI and NtRTI incorporation into areplicating virus genome. Preferably, a dose of an inventive compositiontaken after retroviral exposure is administered within 24 hourssubsequent to the exposure, and more preferably within 12 hourssubsequent to the exposure.

Alternatively, an individual routinely subjected to retroviral exposurecan be protected against the development of a self-replicatingretroviral infection through administration of regular prophylacticdoses of an inventive combination. As a result, an epidemiologicaladvantage exists in controlling the outbreak and spread of a retroviruswithin a population is provided through offering routine doses of aninventive composition prophylactically to high-risk persons such as sexworkers and a short course prophylactic inventive composition touninfected sex trade clientele.

It is appreciated that hybrid dosing regimes of an inventive compositionare also operative herein and include multiple doses prior to retroviralexposure with multiple doses not being administered for a duration orwith sufficient periodicity to arise to the level of a routineprophylactic regime.

The at least one nucleoside reverse transcriptase inhibitor has theattribute of interfering with in vivo viral replication. An NRTIoperative in an inventive prophylactic process includes emtricitabine,lamivudine, zalcitabine, zidovudine, azidothymidine, didanosine,stavudine, abacavir; with the aforementioned specific NRTIs intended toinclude pharmaceutically acceptable salts, esters, ester salts, nitrileoxides, and prodrugs of any of the active agents.

An at least one nucleotide reverse transcriptase inhibitor (NRTI)present in an inventive composition to protect a primate from developinga self-replicating retroviral infection illustratively includestenofovir, adefovir; 2′,3′-dideoxy-3′-fluoroadenisine;2′,3′-dideoxy-3′-fluoroguanasine;3′deoxy-3′-fluoro-5-O-[2-(L-valyloxy)-propionyl]guanosine with theaforementioned specific NtRTIs intended to include pharmaceuticallyacceptable salts, esters, ester salts, nitrile oxides, and prodrugs ofany of the active agents.

Optionally, an inventive composition also includes within an inventivecombination other antiretrovirals such as nonnucleoside reversetranscriptase inhibitors, protease inhibitors, fusion inhibitors, andcombinations thereof. Representative non-nucleoside reversetranscriptase inhibitors operative herein illustratively includedelavirdine, efavirenz, nevirapine, and other diarylpyrimidine (DAPY)derivatives. Representative protease inhibitors operative hereinillustratively include amprenavir, tipranavir, indinavir, saquinavir,lopinavir, ritonavir, fosamprenavir calcium, ritonavir, atazanavirsulfate nelfinavir mesylate, and combinations thereof. An entryinhibitor operative herein as an optional active ingredient in aninventive composition illustratively includes enfuvirtide, Schering C(Schering Plough), S-1360 (Shionogi), and BMS806 (Bristol Myers Squibb).

The dose of individual active components of an inventive prophylacticcomposition is administered to create a therapeutic concentration of theactive composition at the situs of retrovirus initial founder cellpopulation infection prior to viral exposure. It is appreciated thatestablishing a therapeutic concentration at the time of viralreplication for a given NRTI, NtRTI or optional additional active agentin the target cells, includes factors for the therapeutic agent such asthe route of administration, pharmacokinetics, absorption rate based onadministration route, effects of food on oral absorption, in vivodistribution, metabolic pathways, elimination route, race, gender, andage of the subject, single dose incident side effects, long termadministration side effects, and synergistic effects withco-administered active agents. Information related to these factorsconsidered in dosing are available from the United States Food and DrugAdministration (http://www.fda.gov/oashi/aids/virals.html) Preferably,NRTI and NtRTI prophylactic dosing according to the present inventionuses as a starting point the maximal recommended tolerated dosing levelsfor the given active agent combination associated with HAART treatmentprotocols.

An inventive kit is provided that includes a 2-dose package of oraldoses, such as tablets. In an exemplary embodiment of FDA approved NRTIand NtRTIs, each dose contains between 100 and 2500 milligrams (mg) ofemtricitabine and between 100 and 2500 mg of TDF along with instructionsto ingest the first dose approximately 1 to 8 hours prior to potentialretroviral exposure and preferably about 2 hours there before, and asecond dosage to be ingested 20 to 48 hours after potential retroviralexposure, preferably at about 22 hours thereafter. For an adult human,preferably each of the doses includes 200 mg of emtricitabine and 300 mgTDF. A non-human primate dose according to the present invention istypically higher on a mg per kg animal body weight basis by a factortypically ranging from 2 to 10. Additional NRTIs, NtRTIs, NNRTIs,protease inhibitors or entry inhibitors are optionally provided inconcert with either or both of these doses. The kit also includesinstructions as to the timing of doses, contraindications, modificationsassociated with food ingestion, and additional behaviors that therecipient (synonymously described herein as a human primate host) canundertake to reduce the risk of retrovirus exposure and initialinfection. It is also appreciated that a carrier illustrativelyincluding a gel, jelly, cream, ointment, film, sponge, foam,suppository, vaginal ring or other delivery device is providedcontaining an NRTI such as emtricitabine, alone or in combination withan NtRTI such as tenofovir or TDF. The carrier is readily applied tomucosal tissue likely to be exposed to viral transmission as an addedlevel of protection in concert with the oral doses.

An inventive kit is also provided that includes at least one NRTI and atleast one NtRTI compounded as a gel, jelly, cream, ointment, film,sponge, foam, suppository, or applied to a vaginal ring or other likeantiviral barrier. To prepare such a pharmaceutical compounded form, aneffective amount of each of the active agents inclusive of at least oneNRTI and NtRTI is combined in admixture with the pharmaceuticallyacceptable carrier or applied to a surface of the barrier. It isappreciated that the residence time of such a pharmaceutical compositionis maintained at the site of administration through the inclusion of anoptional bioadhesive that provides adhesion to mucosal tissue or thedermis. An inventive composition compounded for application to thedermis or mucosal tissue is provided along with instructions as to thetiming of doses, contraindications, modifications associated with foodingestion, and additional behaviors that the person (synonymouslydescribed herein as a human primate host) can undertake to reduce therisk of retrovirus exposure and initial infection. Optionally, a kitcontaining an oral dosage is combined with a composition compounded forapplication to the dermis, rectal mucosa or vaginal mucosa so as toassure a therapeutically effective combination of NRTI and NtRTI at themucosal point of retroviral entry associated with sexual exposure, aswell as a therapeutically effective serum circulating quantity ofprophylactic antiretrovirals.

The present invention is further detailed with respect to the followingnon-limiting examples. These examples are intended to provide exemplaryspecific embodiments of the present invention and are not intended tolimit the scope of the appended claims.

EXAMPLES Example 1 Antiretroviral Drugs and Doses

A dose of 22 mg/kg of tenofovir disoproxil fumarate (TDF) is givenorally and 20 mg/kg of emtricitabine (FTC) given orally orsubcutaneously to one group of adult male rhesus macaques. The 22 mg/kgTDF dose resulted in an area-under the plasma concentration-time curveover a 24 h interval (AUC) of 4.49 μg×hr/ml which was similar to thevalue of 5.02 μg×hr/ml observed in human receiving 300 mg of TDF. Thedose of 20 mg/kg of FTC resulted in an AUC value (11 μg×hr/ml), alsosimilar to that observed in humans receiving 200 mg of FTC orally(10.0±3.12 μg×hr/ml)⁶. Subcutaneous administration of FTC results inplasma FTC levels comparable to those achieved during oraladministration, indicating a high FTC absorption in rhesus macaques.

Oral administration of FTC and TDF to macaques is by mixing the drugpowders with peanut butter or fruit. Macaques are observed to ensureingestion.

Example 2 Virus Inoculations

A chimeric envelope SHIV_(SF162P3) isolate is used to inoculate themacaques. SHIV_(SF162P3) is a construct that contains the tat, rev, andenv coding regions of HIV-1_(SF162) in a background of SIVmac239. Thisisolate was obtained from the National Institutes of Health (NIH) AIDSResearch and Reference Reagent Program.^(7,8) Virus exposures areperformed 2 hours after drug treatment, and involved non-traumaticinoculation of 1 mL of SHIV_(SF162P3) (10 TCID₅₀ or 7.5×10⁶ viral RNAcopies) into the rectal vault via a sterile gastric feeding tube.⁹Anesthetized macaques remained recumbent for at least 15 min after eachintra-rectal inoculation.

Example 3 SHIV Viral Load Assay

Plasma RNA is quantified using a real-time PCR assay as previouslydescribed.⁵ This assay has a sensitivity of detection of 50 RNAcopies/ml or 10 copies of a pVp1 plasmid carrying the SIVmac239 RT gene.HIV-1 RNA is extracted from 1 mL of plasma using the NucliSensextraction method (bioMérieux). A known amount of virus particles(3×10⁵) from an HIV-1 CM240 virus stock is added to each sample prior toextraction to control for the efficiency of extraction. Reversetranscription is performed using 10 microliters (μl) of extracted RNAand the 2-step TaqMan Gold reverse-transcriptase (RT)-PCR kit (AppliedBiosystems) according to the manufacturer's instructions. PCR reactionsare performed as described using an ABI 7000 Gene Detection System(Applied Biosystems). Virus loads are calculated from a standard curvegenerated with known amount of virus particles. All primers and probesused for SIVmac239 and HIV-1 CM240 have been reported elsewhere.⁵ HIV-1CM240 is obtained from the National Institutes of Health (NIH) AIDSResearch and Reference Reagent Program.

Example 4 Detection of Genotypic Resistance to FTC and Tenofovir

Emergence of FTC and tenofovir resistance is monitored by sequenceanalysis of SIV RT (551 bp; amino acids 52 to 234) and by a moresensitive allele-specific real-time PCR method for the K65R and M184Vmutations. Sequence analysis was done from plasma viruses using anRT-PCR procedure as previously described.⁵ The Vector NTI program(Version 7, 2001) is used to analyze the data and to determine deducedamino-acid sequences. Detection of low frequency of K65R and M184Vmutants in plasma by real-time PCR is performed as previouslydescribed.¹⁰ These assays have a detection limit of 0.4% of K65R and0.6% of M184V cloned sequences in a background of wild type plasmid.

Example 5 Virus-Specific Antibody Responses

Virus-specific serologic responses (IgG and IgM) are measured using asynthetic-peptide EIA (Genetic Systems HIV-1/HIV-2) assay.

Example 6 Statistical Methods

The exact log-rank test is used for a discrete-time survival analysis ofthe treatment and control groups, with use of the number of inoculationsas the time variable. The Cox proportional hazards model is used toestimate the relative hazard ratio (HR). Percent protection iscalculated from the HR value using the formula: (1−1/HR)×100. Allstatistical analyses for calculation of the efficacy of the differentinterventions are performed using SAS software (version 9.1; SASInstitute) and StatXact software (version 6.3; Cytel).

Example 7 Routine Dosing Experimental Design

Macaques are exposed rectally once weekly for up to 14 weeks toSHIV162p3 which contains an R5 tropic HIV-1 envelope that resemblesnaturally transmitted viruses. The SHIV162p3 challenge dose is 10 TCID₅₀or 7.6×10⁵ RNA copies which is similar to HIV-1 RNA levels in semenduring acute infection in humans.¹¹ Virus exposures are terminated whena macaque became infected. FIG. 1 shows the study design and theinterventions evaluated in each group of macaques. Three prophylacticdrug treatments of increasing drug potency are each given once daily toa group of six macaques. Animals in Group 1 were treated subcutaneouslywith 20 mg/kg of FTC alone. Animals in Group 2 received orally acombination of FTC (20 mg/kg) and TDF (22 mg/kg). Animals in Group 3 hadthe most protective treatment with subcutaneous 20 mg/kg of FTC and a 22mg/kg of tenofovir (PMPA). The rate of infection in each group iscompared with that seen in 18 untreated control macaques (9 real timeand 9 historical controls).

All treated macaques received the corresponding drugs 7 to 9 days priorto the first virus inoculation to achieve steady-state plasma levels.Treated animals that remained uninfected during the 14 challengesreceived 28 days of post-exposure prophylaxis after the last challenge.Protection was defined as absence of persistent viremia andseroconversion. Treated animals that became infected continued treatmentfor an average of 21 weeks (range=13 to 29) to monitor for plasmaviremia and drug resistance development.

Example 8 Survival Curves

FIG. 2 shows the survival curves observed for each group of animals perExample 7. Data with TDF (20 mg/kg) is also provided for comparison.Untreated macaques are infected after a median of 2 rectal exposures(mean=4). The majority of the animals (13/18 or 72%) are infected duringthe first 4 challenges (median=2); 4 (22%) are infected betweenexposures 8 and 14 (mean=10), and only 1 (6%) remained uninfected after14 exposures. The median 2 exposures for infection in controls suggeststhat an animal receiving prophylactic treatment and remaining uninfectedafter 14 virus challenges would have been protected against a median of7 rounds of transmissions. Treatments of Groups 1-3 are all protectiveto a degree with a clear dose-response relationship being observed. All6 macaques in Group 3 that received the most potent inventivecomposition remained uninfected demonstrating that full protectionagainst repeated challenges is possible. Of the 6 macaques in Group 2, 4were protected and only 2 (animal reference numbers AI-54 and AG-81)became infected at exposures 9 and 12. Compared to controls, infectionin this group is reduced by 7.8-fold (Cox proportional hazard ratio[HR]=7.8, p=0.0075). Infection in both animals is significantly delayedcompared to the untreated controls (p=0.0004). These 2 macaques becameseropositive 2 weeks after the first detectable viral RNA in plasma andboth were proviral DNA positive at weeks 10 and 12, respectively. Of the6 macaques in Group 1 receiving FTC only, 2 remained protected after 14exposures and 4 had the first detectable viral RNA at exposures 5(AG-80), 10 (AG-46), 12 (AH-04), and 13 (AG-07), respectively. Survivalanalysis showed a statistically significant difference from untreatedcontrols (p=0.004). Compared to controls, infection is reduced 3.8-foldmacaques (Cox proportional hazard ratio [HR]=3.8, p=0.021). Infection inthese 4 animals is also confirmed by PCR amplification of proviral DNAfrom PBMCs and by serology; antibody responses are detectable 3, 1, 2,and 6 weeks after the first detectable RNA, respectively. FIG. 2 alsoshows that the protection achieved with FTC alone was higher than thatpreviously seen in 4 animals receiving TDF,⁵ consistent with theslightly higher potency of FTC, although the difference was notstatistically significant (p=0.5).

Example 9 Prophylactic Breakthrough Infections and Drug ResistanceEmergence

Since the dynamics of breakthrough infections that occur duringinventive prophylaxis and drug resistance emergence are unknown, the 6infected animals from Groups 1 and 2 are followed under continued drugtreatment. FIG. 3 compares the virus load kinetics in the 6 breakthroughinfections with those in 12 untreated macaques that had sufficientfollow-up samples. The mean peak viremia in the 6 treated macaques was4.9±0.5 log₁₀ RNA copies/ml, 2.0 log₁₀ lower than in untreated controls(6.9±0.3 log₁₀ RNA). FIG. 3 also shows that such differences in viremiawere maintained up to week 11 as indicated by similar rate of virus loaddecline seen in the two groups of animals (−0.23±0.02 log₁₀/week intreated vs. −0.29±0.02 log₁₀/week in untreated controls). The individualvirus load kinetics in the 6 breakthrough infections are shown in FIG.4. Three FTC (AG-80, AH-04, and AG-07) and one of the FTC/TDF (AG-81)failures had undetectable virus loads 3, 4, 7, and 11 weeks after thepeak in viremia, respectively; viremia in these animals remainedconsistently low or undetectable for up to 20 weeks. In contrast, all 12untreated macaques had detectable virus loads during a median follow-upperiod of 7 weeks (range=5-36 weeks). The arrow in FIG. 4 denotes thefirst detectable antibody response. Grey circles indicate detectableM184V/I mutation; wild type sequences are shown in black full circles.Open circles are provided for data points not genotyped.

Drug resistance testing showed that wild type virus initiated all 6breakthrough infections in Groups 1 and 2 reflecting residual virusreplication in target cells not protected by drugs (FIG. 4). Fouranimals had no evidence of drug resistance despite extended treatment(median=23 weeks). Only 2 animals had detectable M184V (AG-46,FTC-treated) or M184I (AI-54 FTC/TDF-treated) mutations associated withFTC resistance at week 4 and 10, respectively. The tenofovir-associatedK65R mutation is not detected in the 2 Group 2 animals receivingFTC/TDF. FIG. 4 also shows that the 2 macaques that selected M184V/I hadthe highest peak viremias. Without intending to be bound to a particulartheory, it is hypothesized that more virus replication in these animalsmay have facilitated drug resistance selection. Reductions in acuteviremia are proposed to contribute at a population level to a decreasein virus transmissibility.

Example 10 Single Dosing

The process of Example 7 is repeated in Group 3 with drugs only beingadministered 2 hours prior to and 22 hours subsequent to eachinoculation. The resultant survival curves are comparable to thosedetailed in Example 8.

Example 11 Single Dosing with Suppository

A group of 6 macaques received the drug treatment of Group 3 per Example7 in the form of a gel inserted rectally containing 300 mg of tenofovirand 300 mg lamuvidine (3-TC) 1 hour before viral inoculation withobservation to assure that the suppository is not voided. The gel isformed by compounding tenofovir and 3-TC in 2% by weight hydroxyethylcellulose (HEC)-based gel in both a vaginal formulation (pH 4.5) andrectal formulation (pH 6.5) containing (w/v) 3% tenofovir, and 3% 3-TC.The gels are stable at room temperature for at least five months with noloss in activity; and gels retained full activity at both pH 4.5 and pH6.5 at levels equivalent to those observed for tenofovir and 3-TCpreparations in water. Using an MT4/MTT phenotypic assay, all gels weretested for activity against wild-type HIV-1_(IIXB2), and resistant HIV-1viruses containing the K65R or M184V mutations. No significantcytotoxicity is seen in the cervical explant model.

Viral protection of the macaques is maintained throughout the study.

REFERENCES CITED

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Patent documents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. These documents and publications are incorporatedherein by reference to the same extent as if each individual document orpublication was specifically and individually incorporated herein byreference.

The foregoing description is illustrative of particular embodiments ofthe invention, but is not meant to be a limitation upon the practicethereof. The following claims, including all equivalents thereof, areintended to define the scope of the invention.

1. A method, comprising: prohibiting a primate subject from acquiring animmunodeficiency retrovirus infection by: a. determining if the primatesubject is negative for a detectable immunodeficiency retrovirusinfection; and b. orally administering a plurality of doses of at leastone tablet comprising 200 mg emtricitabine (FTC) and a pharmaceuticallyeffective amount of a tenofovir prodrug to the subject if the primatesubject is negative for the immunodeficiency retrovirus infection,wherein at least a first dose occurs before the primate subject ispotentially exposed to the immunodeficiency retrovirus; and testing theprimate subject after the oral administration to confirm that thesubject has remained negative for the detectable immunodeficiencyretrovirus infection following a plurality of doses.
 2. The method ofclaim 1, wherein the tenofovir prodrug is tenofovir disoproxil fumaratein an amount of 300 mg.
 3. The method of claim 1, wherein orallyadministering the at least one tablet comprises orally administering onetablet per day.
 4. The method of claim 1, wherein the tenofovir prodrugcomprises a tenofovir ester.
 5. The method of claim 1, wherein the oraladministration of the at least one tablet results in an area under theplasma concentration-time curve (AUC) over a 24 hour interval foremtricitabine in the primate subject of 6.88 μg·hr/mL to 13.12 μg·hr/mL.6. The method of claim 1, wherein the oral administration of the atleast one tablet results in an area under the plasma concentration-timecurve (AUC) over a 24 hour interval for emtricitabine in the primatesubject of 11 μg·hr/mL.
 7. The method of claim 1, wherein the testingthe primate subject comprises a polymerase chain reaction (PCR) assay todetect the presence of the immunodeficiency retroviral genome in theprimate subject.
 8. The method of claim 1, wherein the oraladministration of the at least one tablet results in an absence of animmunodeficiency retrovirus seroconversion in the primate subject. 9.The method of claim 1, wherein the primate subject is an adult humansubject.
 10. The method of claim 1, wherein the oral administration ofthe plurality of doses of the at least one tablet comprises a treatmentregimen over a time period, and wherein the testing of the primatesubject occurs regularly during the time period of the treatmentregimen.
 11. The method of claim 1, wherein the oral administration ofthe plurality of doses of the at least one tablet prohibits a subsequentHIV-1 exposure of the primate subject from becoming a self-replicatingHIV-1 infection.
 12. A method, comprising inhibiting a human subject whois uninfected with human immunodeficiency virus 1 (HIV-1) from becominginfected, by: a. screening the human subject to confirm that the subjectdoes not have a detectable HIV-1 infection; b. orally administering aplurality of doses of at least one tablet comprising 200 mgemtricitabine (FTC) and a pharmaceutically effective amount of a prodrugof tenofovir to the subject if the primate subject is confirmed to nothave an HIV-1 infection, wherein the administration occurs before thehuman subject is exposed to HIV-1; and c. testing the human after thesubject has been potentially exposed to HIV-1 to confirm that the humansubject does not have an HIV-1 infection.
 13. The method of claim 12,wherein the prodrug of tenofovir is tenofovir disoproxil fumarate in anamount of 300 mg.
 14. The method of claim 12, wherein the prodrug oftenofovir is a tenofovir ester.
 15. The method of claim 12, whereinadministration of a single dose of the tablet results in an area underthe plasma concentration-time curve (AUC) for emtricitabine of about6.88 μg·hr/mL to about 13.12 μg·hr/mL.
 16. The method of claim 12,wherein administration of a single dose of the tablet results in an areaunder the plasma concentration-time curve (AUC) for emtricitabine of 11μg·hr/mL.
 17. The method of claim 12, wherein the oral administration isa daily oral dose of the tablet.
 18. The method of claim 17, wherein thedaily oral dose of the tablet inhibits a subsequent HIV-1 exposure ofthe human subject from becoming a self-replicating HIV-1 infection. 19.The method of claim 17, wherein the daily oral dose of the tablet isadministered daily for several months, and wherein the subject is testedfor HIV-1 infection during the course of the several months ofadministration.
 20. The method of claim 19, wherein the daily oral doseof the tablet results in steady-state plasma levels of emtricitabine andtenofovir in the primate subject.
 21. A pre-exposure prophylactictreatment method for preventing an exposure of a human subject to humanimmunodeficiency virus 1 (HIV-1) from becoming a HIV-1 self-replicatinginfection, comprising: a. confirming that the human subject isserologically negative for HIV-1; b. orally administering a plurality ofdaily doses of a tablet comprising 200 mg emtricitabine (FTC) and apharmaceutically effective amount of a tenofovir prodrug to the humansubject if the human subject is serologically negative for HIV-1,wherein the administration is initiated before the human subject hasbeen exposed to HIV-1; and c. testing the human subject after severalmonths of the oral administration to confirm that the subject does nothave a self-replicating HIV-1 infection.
 22. The treatment method ofclaim 21, wherein the tenofovir prodrug is tenofovir disoproxil fumaratein an amount of 300 mg.
 23. The treatment method of claim 21, whereinthe tenofovir prodrug is a tenofovir ester.
 24. The treatment method ofclaim 23, wherein if the testing determines that the subject does nothave a self-replicating HIV-1 infection after the several months ofdaily doses of the tablet, then continuing with the daily doses of thetablet.
 25. The method of claim 23, wherein the oral administration ofthe daily doses of the tablet results in the human subject beingprotected from developing a self-replicating HIV-1 infection followingsexual exposure to an HIV-1-infected subject.
 26. A pre-exposureprophylactic treatment method for preventing an exposure of a humansubject to human immunodeficiency virus 1 (HIV-1) from becoming a HIV-1self-replicating infection, comprising: a. confirming that the humansubject is negative for an HIV-1 infection; b. orally administering adaily dose of a tablet comprising 200 mg emtricitabine (FTC) and atenofovir prodrug to the human subject for several months if the humansubject is serologically negative for HIV-1; and c. testing the humansubject after the several months of the oral administration to confirmthat the human subject has not developed a self-replicating retroviralinfection.
 27. The method of claim 26, wherein the tenofovir prodrug istenofovir disoproxil fumarate in an amount of 300 mg.
 28. The method ofclaim 26, wherein the tenofovir prodrug is a tenofovir ester.
 29. Themethod of claim 26, wherein the oral administration of the tabletresults in an area under the plasma concentration-time curve (AUC) overa 24 hour interval for emtricitabine in the human subject of 10.0±3.12μg·hr/mL.
 30. The method of claim 26, wherein the oral administration ofthe tablet results in an area under the plasma concentration-time curve(AUC) over a 24 hour interval for emtricitabine in the human subject of11 μg·hr/mL.